Chemically synthesized oligonucleotides have been used in hybridization assays for some time, and by now are fairly routine. However, for uses which imitate biological processes, e.g., hybridizations of nucleic acid probes on a template followed by ligation, the normal 5' hydroxyl terminus must be converted to a phosphate to provide the proper substrate for a ligase. Methods of phosphorylating include enzymatic and synthetic as described below. The present invention describes a particular synthetic method, wherein silyl substituted alcohols are useful reagents.
Synthesis of silyl substituted alcohols has been previously achieved by oxidation of organoboranes. The organoboranes are in turn prepared by the Grignard reaction or by hydroboration of vinyl- and allyl-silanes. This technique is described in Kumada, et al. J. Organometal. Chem. 6:490-495 (1966) and Seyferth, J. Am. Chem. Soc. 81:1844 (1959). This technique is useful only when the requisite vinyl or allylsilanes can be synthesized or obtained commercially. However, if the desired vinyl silane is commercially unavailable or difficult to synthesize this method is not useful.
Alpha silyl esters have been prepared by reacting a chlorosilane and an alpha-bromo ester with zinc under Reformatsky conditions. See Feesenden, et al. J. Org. Chem. 32:3535 (1967).
An important drawback of these synthesis methods is the side reactions which can occur leading to undesirable products and decreasing the yields. In conventional processes for hydrolyzing silyl substituted esters to the corresponding silyl substituted alcohol, a carbanion intermediate is generally formed. With .beta.-silyl substituted alcohols, fragmentation to the silanol and an olefin can occur; with alpha silyl substituted alcohols, a Brook rearrangement to give a silyl protected ether will occur. Thus, in these carbanion intermediates there is a strong tendency for an elimination reaction whereby the silicon atom shifts to the oxygen atom to form the R.sub.3 SiOH byproduct. This tendency is especially pronounced when the reaction is performed in strong base and when groups substituted on the silicon are particularly bulky.
Hydrosilation, the addition of H and silyl compounds across the double bond of an olefin, has also been described in the literature. See Collman, et al. Principles and Applications of Organotransition Metal Chemistry, University Science Books (1980) p. 384-389 and Pegram, et al. Carbohydrate Research 184:276 (1988). In a particularly relevant hydrosilation reaction, Salimgareeva, et al., Zh. Obshch. Khim 48(4):930-31 (1978)(Russian) (see also C.A. 89:146961y) report hydrosilation of vinyl acetate with dimethylsilane. This reaction resulted in two silyl substituted products: a monoacetate and a diacetate. The reference fails to describe synthesis of any silyl alcohol or the use of synthesis of any bulky silyl substituted compound.
Honda, et al. Tetrahedron Letters, 22(22): 2093-2096 (1981) describe a .beta.-silyl substituted ethanol wherein the silyl group bears two phenyl and one methyl substituent. Honda, et al. used this compound to prepare a phosphorylating agent which places a protected phosphate group between nucleotides in oligonucleotide synthesis. The substituted silyl protecting group can be removed to give a silyl fluoride compound, ethylene and phosphate. The substituted silyl ethanol was obtained by reduction of the bisphenylmethyl silyl acetate with LiAlH.sub.4 according to a modification of the procedure of Gerlach, Helv Chim. Acta, 60:3039 (1977).
Other silyl substituted ethanols have been described in the literature, but primarily include alkyl substituted silyl groups. Examples of such silyl ethanols and their literature citations are found in the following table.
TABLE 1 ______________________________________ ##STR1## R R' R" Literature Citation ______________________________________ isopropyl isopropyl isopropyl CA111(11):97352n methyl methyl propenyl CA105(13):115112s CA108(17):150554w methyl n-butyl n-butyl CA88(3):23391j: CA83(11):97563k; CA78(15):93640g; & CA78(13):84526x methyl methyl t-butyl CAOLD (prior to 1967) ethyl ethyl ethyl CA111(11):973552n; CA98(9):72207u; CA87(15):117488c; CA85(21):154709e; CA80(11):59132z; & CA77(18):120049a propyl propyl propyl CA103(19):160573n phenyl phenyl methyl Honda, et al., Tetrahedron Letters, 22(22):2093-2096 (1981) ______________________________________
Triphenyl silane (not the alcohol) has been described by Lesage, et al. J. Org. Chem 55:5413 (1990) as a useful reducing agent.
In addition to the method of Honda, et al. (See above), several methods for phosphorylating the 5' terminus of an oligonucleotide are known. Initially, enzymatic methods using polynucleotide kinase were employed after the oligonucleotide was synthesized and removed from the solid support. Others have taught methods and reagents for chemically phosphorylating a synthesized oligonucleotide prior to its removal from the solid support. Some of these are described below.
Kondo, et al. Nucl. Acids Res. Symposium Series 16:161-164 (1985) describe phosphotriester (1) and phosphoramidite (2) reagents for phosphorylating 5' termini. Phosphorylation is achieved by preparing a special diphosphorylated (3'-5') nucleotide which is added as the last nucleotide in the chain. The 3' phosphate is linked via the phosphotriester or phosphoramidite to the extending nucleotide chain. The 5' phosphate is protected with a protecting group which is ultimately removed.
Uhlmann, et al. Tetrahedron Letters 27(9): 1023-1026 (1986) describe a phosphoramidite phosphorylating reagent using a p-nitrophenylethyl group as a blocking group. They mention that the hydrophobic p-nitrophenylethyl is advantageous in that phosphorylated compounds can be separated from non-phosphorylated compounds by reversed phase HPLC.
Uhlmann, et al, however, used only hexamers to which the p-nitrophenylethyl "handle" was attached. A similar approach using a p-nitrophenylethyl handle with 20-mers is described by G. Zon in chapter 14 of HPLC in Biotechnology, (W. S. Hancock, ed), J. Wiley & Sons, New York, N.Y. pp. 359-363 (1990). The purification results obtained by Zon with this method are marginal.
Marugg, et al. Nucl. Acids Res. 12(22):8639-8651 (1984) describe a new phosphorylating agent, 2-cyano-1,1-dimethylethoxy dichlorophosphine. This agent has the alleged advantage of being removed under just basic conditions.
Himmelsbach, et al. Tetrahedron Letters 23(46):4793-4796 (1982) describe a new phosphorylating agent, bis-(p-nitrophenylethyl) phosphoromonochloridate. Van der Marel, et al. Tetrahedron Letters, 22(19):1463-1466 (1981) describe a morpholino phosphoro bis-3-nitro-1, 2, 4-triazolidate.
Horn, et al. Tetrahedron Letters 27 (39):4705-4708 (1986) describe a phosphorylating reagent including a 4, 4'dimethoxytrityl group which, upon release, can be used to monitor the efficiency of phosphorylation. This disclosure appears to be quite similar to that of EP-A-304 215 and to the commercially available Ciontech product known as 5' Phosphate-On.
Lipshutz, et al. Tetrahedron Letters 30(51): 7149-7152 (1989) ("Lipshutz 1989") and Lipshutz, et al. Tetrahedron Letters 21:3343-3346 (1980) ("Lipshutz 1980") and Von Peter Sieber, Helvetica Chimica Acta 60:2711 (1977) all disclose the use of fluoride in the removal of a silyl protecting group. In this regard, they are similar to Honda, et al. (See above).
While each of the above reagents and methods are adequate for phosphorylating synthesized oligonucleotides, each has draw backs as well. For example, each of the recited references discloses a method for removing the phosphate blocking group to generate the native 5' phosphate. Some (e.g. Horn, et al.) describe a blocking agent having a detectable characteristic (e.g. color) by which the extent of phosphorylation can be monitored. While the extent of phosphorylation can be monitored by this means, it provides no means for purification. Uhlmann, et al. suggest that the hydrophobic p-nitrophenylethyl group can be used prior to cleavage to separate phosphorylated hexamers by HPLC. The protected hexamers cited by Uhlmann, having a relatively low molecule/protecting group mass ratio, are generally too short to provide specificity necessary in hybridization assays.
However, none of the references teach phosphorylating/blocking reagents comprising silyl substitutes. Further, none suggest that the silyl protecting group can be used to purify phosphorylated nucleotides from unphosphorylated failure product. The present invention seeks to overcome these disadvantages.